Natural products that demonstrate pharmacological efficacy are very important chemical scaffolds for drug design. While the vast majority of small molecule natural products will eventually be screened for biological activity, only a small fraction of these drug-like agents will move forward in the drug development pipeline. Currently, a considerable amount of effort is being directed through high throughput screening to select lead candidates based on their ability to bind to validated disease-associated protein targets.
A new approach has emerged that employs biologically active small molecules as cell permeable probes to identify novel binding targets and to study their function. Since pharmacological activity of a small molecule resides principally through binding interaction with its hypothetical biological target(s) (e.g., protein receptor), it is the discovery of such a protein receptor is of critical importance in drug development. Furthermore, the identification of the specific amino acid of the protein target to which the small molecule binds offers molecular insight into the specificity of such binding interactions. Consequently, the use of the binding site information allows one to look for similar binding sites in other classes of proteins, molecular information that helps expand the target cell types/organisms and clinical indications for the small molecule drug lead. The discovery of VIAGRA™ for male sexual dysfunction is one such example where research on phoshodiesterase inhibitors for cardiovascular dysfunction led to the serendipitous identification of a novel therapeutic area for this class of drugs.
In small molecule protein target discovery research, the most critical element is the innovative chemical design to convert a drug-like chemical compound into a useful biologically active chemical probe that affords the ability to use the reagent for identification of its in vivo protein binding target. Careful consideration is paid to not alter any of the small molecule's biological activities when the synthetic analog is generated. In addition, it is also important that a level of specificity for protein binding is demonstrated. Chemical radioisotope tagging is commonly used to generate a radiolabeled analog of the small molecule to help in protein target detection. However, a major drawback with this approach is that this method does not afford a direct means to isolate the binding target of this agent.
One recent screening method uses affinity tagging small molecules with biotin to form a small molecule analog, which seemingly provides the potential for isolating a small molecule binding target. Unfortunately, current techniques of biotin affinity tagging have not realized the full potential of isolating the binding target for small molecule agents due, in part, to the presence of the biotin attached to the small molecule, which may interfere with the natural binding of the small molecule analog with its target.
One small molecule is the natural product Withaferin A (hereinafter “WFA”), which is a potent angiogenesis inhibitor that targets the ubiquitin-proteasome pathway in vascular endothelial cells. WFA is an important prototype of the withanolide class of natural products and is a highly oxygenated steroidal lactone that is found in the medicinal plant Withania somnifera and its related solanaceas species. The withanolides are known to exert very potent and diverse cytotoxic, anti-stress, cardioactive, central nervous system, and immunomodulatory activities. Since the early discovery of WFA during the 1960s, the major interest has been on its anti-tumor cytotoxic activities. The mesenchymal type-III intermediate filament (“IF”) protein vimentin plays a critical role in wound healing, angiogenesis and cancer growth.
The role of vimentin in human and animal disorders, including those disorders associated with aberrant or elevated expression of vimentin, and diseases associated with angiogenesis are known in the prior art, e.g. by the following references, U.S. Pat. Nos. 5,716,787; 5,932,545; 6,846,841; 7,132,245; 7,175,844; U.S. Patent application publication no. 2006/0014225; Hertig A, Verine J, Mougenot B, Jouanneau C, Ouali N, Sebe P, Glotz D, Ancel P Y, Rondeau E, Xu-Dubois Y C; Risk factors for early epithelial to mesenchymal transition in renal grafts, Am J Transplant; 2006 December; 6(12):2937-46 (hereinafter “Hertig et al.”); Kenyon, B. M., et al., “Effects of Thalidomide and Related Metabolites in a Mouse Corneal Model of Neovascularization”, Exp. Eye Res., 64:971-978 (1997) (hereinafter “Kenyon et al.”); Cole, C. H., et al., “Thalidomide in the management of chronic graft-versus-host disease in children following bone marrow transplantation”, Bone Marrow Transplantation, 14:937-942 (1994) (hereinafter “Cole et al.”); Russell, M. E., et al., “Chronic Cardiac Rejection in the LEW to F344 Rat Model”, J. Clin. Invest., 97(3):833-838 (1996) (hereinafter “Russell et al.”; Kwon Y S, Kim J C. Inhibition of corneal neovascularization by rapamycin, Exp Mol Med. 2006 Apr. 30; 38(2):173-9 (hereinafter “Kwon et al.”); Zogakis T G, Libutti S K, General aspects of anti-angiogenesis and cancer therapy, Expert Opin Biol Ther, 2001 March; 1(2):253-75, Review; Kirsch M, Santarius T, Black P M, Schackert G, Therapeutic anti-angiogenesis for malignant brain tumors. Onkologie, 2001 October; 24(5):423-30. Review hereinafter “Zogakis et al.”); Manzano R, Peyman G, Khan P, Carvounis P, Kivilcim M, Ren M, Lake J, Chevez-Barrios P, Inhibition of experimental corneal neovascularization by Bevacizumab(AVASTIN), Br J Opthalmol. 2006 Dec. 19 (hereinafter “Manzano et al.”); Yeh J R, Mohan R, Crews C M, The antiangiogenic agent TNP-470 requires p53 and p21 CIP/WAF for endothelial cell growth arrest, Proc Natl Acad Sci USA. 2000 Nov. 7; 97(23):12782-7 (hereinafter “Yeh et al.”); Yu Y, Moulton K S, Khan M K, Vineberg S, Boye E, Davis V M, O'Donnell P E, Bischoff J, Milstone D S, E-selectin is required for the antiangiogenic activity of endostatin, Proc Natl Acad Sci U S A. 2004 May 25; 101(21):8005-10 (hereinafter “Yu et al.”); Murthy R C, McFarland T J, Yoken J, Chen S, Barone C, Burke D, Zhang Y, Appukuttan B, Stout J T, Corneal transduction to inhibit angiogenesis and graft failure, Invest Opthalmol V is Sci. 2003 May; 44(5):1837-42 (hereinafter Murthy et al.”); Rogers M S, Rohan R M, Birsner A E, D'Amato R J. Genetic loci that control the angiogenic response to basic fibroblast growth factor. FASEB J. 2004 July; 18(10):1050-9 (hereinafter “Rogers et al.”); Zhang M, Volpert O, Shi Y H, Bouck N. Maspin is an angiogenesis inhibitor, Nat. Med. 2000 February; 6(2):196-9 (hereinafter “Zhang et al.”); and Mor-Vaknin N, Punturieri A, Sitwala K, Markovitz D M, Vimentin is secreted by activated macrophages, Nat Cell Biol. 2003 January; 5(1):59-63 (hereinafter “Mor-Vaknin et al.”); all herein incorporated by reference.
Although the role of vimentin, and diseases associated with angiogenesis are known in the prior art, the non-cytotoxic anti-inflammatory and immunomodulatory mechanisms of WFA have thus far remained rather poorly characterized. These latter disease-altering activities are highly pertinent to the practice of ayurveda, a traditional form of Indian medicine, which has borne out many effective formulations from W. somnifera, especially for the treatment of chronic human diseases such as arthritis and female bleeding disorders.